LOCUS       sequence-206009-        7779 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac vector for sgRNA cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7779)
  AUTHORS   Ishida K, Xu H, Sasakawa N, Lung MSY, Kudryashev JA, Gee P, Hotta A
  TITLE     Site-specific randomization of the endogenous genome by a 
            regulatable CRISPR-Cas9 piggyBac system in human cells.
  JOURNAL   Sci Rep. 2018 Jan 10;8(1):310. doi: 10.1038/s41598-017-18568-4.
  PUBMED    29321585
REFERENCE   2  (bases 1 to 7779)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2018-01-10"; volume: "8(1):310. doi"; pages: " 
            10.1038/s41598-017-18568-4"
COMMENT     Sequence Label: pPV-H1-ccdB-mEF1-alpha-RiH
FEATURES             Location/Qualifiers
     source          1..7779
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          188..1135
                     /label=mEF-1-alpha intron
                     /note="intron upstream of the start codon of mouse 
                     EF-1-alpha"
     CDS             1187..1864
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             1187..1864
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(1317..1335)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1585..1604
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1779..1802
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    1873..2317
                     /label=FMDV IRES
                     /note="internal ribosome entry site (IRES) of the 
                     foot-and-mouth disease virus"
     promoter        2351..2397
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        2389..2407
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2417..3442
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     repeat_region   4225..4259
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4352..4369)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4523..5111)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4603..4622)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5282..6073,6074..6142))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5905..5924
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6143..6247)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   6304..6366
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        6710..6924
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     6845..6864
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     CDS             7262..7567
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     7446..7465
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(7608..7732)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gctctagact agtgcggccg cccctccccc acagcagggg cggggtcccg cgcccaccgg
       61 aaggagcggg ctcggggcgg gcggcgctga ttggccgggg cgggcctgac gccgacgcgg
      121 ctataagaga ccacaagcga cccgcagggc cagacgttct tcgccgaagc ttgccgtcag
      181 aacgcaggtg aggggcgggt gtggcttccg cgggccgccg agctggaggt cctgctccga
      241 gcgggccggg ccccgctgtc gtcggcgggg attagctgcg agcattcccg cttcgagttg
      301 cgggcggcgc gggaggcaga gtgcgaggcc tagcggcaac cccgtagcct cgcctcgtgt
      361 ccggcttgag gcctagcgtg gtgtccgcgc cgccgccgcg tgctactccg gccgcactct
      421 ggtctttttt ttttttgttg ttgttgccct gctgccttcg attgccgttc agcaataggg
      481 gctaacaaag ggagggtgcg gggcttgctc gcccggagcc cggagaggtc atggttgggg
      541 aggaatggag ggacaggagt ggcggctggg gcccgcccgc cttcggagca catgtccgac
      601 gccacctgga tggggcgagg cctggggttt ttcccgaagc aaccaggctg gggttagcgt
      661 gccgaggcca tgtggcccca gcacccggca cgatctggct tggcggcgcc gcgttgccct
      721 gcctccctaa ctagggtgag gccatcccgt ccggcaccag ttgcgtgcgt ggaaagatgg
      781 ccgctcccgg gccctgttgc aaggagctca aaatggagga cgcggcagcc cggtggagcg
      841 ggcgggtgag tcacccacac aaaggaagag ggcctggtcc ctcaccggct gctgcttcct
      901 gtgaccccgt ggtcctatcg gccgcaatag tcacctcggg cttttgagca cggctagtcg
      961 cggcgggggg aggggatgta atggcgttgg agtttgttca catttggtgg gtggagacta
     1021 gtcaggccag cctggcgctg gaagtcattt ttggaatttg tccccttgag ttttgagcgg
     1081 agctaattct cgggcttctt agcggttcaa aggtatcttt taaacccttt tttaggtgtt
     1141 gtgaaaacca ccgctaattc aaagcaaccg gtgatatcgg atcaccatgg cctcctccga
     1201 ggacgtcatc aaggagttca tgcgcttcaa ggtgcgcatg gagggctccg tgaacggcca
     1261 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     1321 gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     1381 ccagtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     1441 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     1501 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     1561 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     1621 ctccaccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agatgaggct
     1681 gaagctgaag gacggcggcc actacgacgc cgaggtcaag accacctaca tggccaagaa
     1741 gcccgtgcag ctgcccggcg cctacaagac cgacatcaag ctggacatca cctcccacaa
     1801 cgaggactac accatcgtgg aacagtacga gcgcgccgag ggccgccact ccaccggcgc
     1861 ctaataccta ggagcaggtt tccccaatga cacaaaacgt gcaacttgaa actccgcctg
     1921 gtctttccag gtctagaggg gtaacacttt gtactgcgtt tggctccacg ctcgatccac
     1981 tggcgagtgt tagtaacagc actgttgctt cgtagcggag catgacggcc gtgggaactc
     2041 ctccttggta acaaggaccc acggggccaa aagccacgcc cacacgggcc cgtcatgtgt
     2101 gcaaccccag cacggcgact ttactgcgaa acccacttta aagtgacatt gaaactggta
     2161 cccacacact ggtgacaggc taaggatgcc cttcaggtac cccgaggtaa cacgcgacac
     2221 tcgggatctg agaaggggac tggggcttct ataaaagcgc tcggtttaaa aagcttctat
     2281 gcctgaatag gtgaccggag gtcggcacct ttcctttgca attactgacc ctatgaatac
     2341 aactgactgt ttgacaatta atcatcggca tagtatatcg gcatagtata atacgactca
     2401 ctataggagg gccaccatga agaaacctga actgacagca acttctgttg agaagtttct
     2461 cattgaaaaa tttgattctg tttctgatct catgcagctg tctgaaggtg aagaaagcag
     2521 agccttttct tttgatgttg gaggaagagg ttatgttctg agggtcaatt cttgtgctga
     2581 tggtttttac aaagacagat atgtttacag acactttgcc tctgctgctc tgccaattcc
     2641 agaagttctg gacattggag aattttctga atctctcacc tactgcatca gcagaagagc
     2701 acaaggagtc actctccagg atctccctga aactgagctg ccagctgttc tgcaacctgt
     2761 tgctgaagca atggatgcca ttgcagcagc tgatctgagc caaacctctg gatttggtcc
     2821 ttttggtccc caaggcattg gtcagtacac cacttggagg gatttcattt gtgccattgc
     2881 tgatcctcat gtctatcact ggcagactgt gatggatgac acagtttctg cttctgttgc
     2941 tcaggcactg gatgaactca tgctgtgggc agaagattgt cctgaagtca gacacctggt
     3001 ccatgctgat tttggaagca acaatgttct gacagacaat ggcagaatca ctgcagtcat
     3061 tgactggtct gaagccatgt ttggagattc tcaatatgag gttgccaaca tttttttttg
     3121 gagaccttgg ctggcttgca tggaacaaca aacaagatat tttgaaagaa gacacccaga
     3181 actggctggt tcccccagac tgagagccta catgctcaga attggcctgg accaactgta
     3241 tcaatctctg gttgatggaa actttgatga tgctgcttgg gcacaaggaa gatgtgatgc
     3301 cattgtgagg tctggtgctg gaactgttgg aagaactcaa attgcaagaa ggtctgctgc
     3361 tgtttggact gatggatgtg ttgaagttct ggctgactct ggaaacagga gaccctccac
     3421 aagacccaga gccaaggaat gaatattagc tagattatcc ctaatacctg ccaccccact
     3481 cttaatcagt ggtggaagaa cggtctcaga actgtttgtt tcaattggcc atttaagttt
     3541 agtagtaaaa gactggttaa tgataacaat gcatcgtaaa accttcagaa ggaaaggaga
     3601 atgttttgtg gaccactttg gttttctttt ttgcgtgtgg cagttttaag ttattagttt
     3661 ttaaaatcag tactttttaa tggaaacaac ttgaccaaaa atttgtcaca gaattttgag
     3721 acccattaaa aaagttaaat gagaaacctg tgtgttcctt tggtcaacac cgagacattt
     3781 aggtgaaaga catctaattc tggttttacg aatctggaaa cttcttgaaa atgtaattct
     3841 tgagtttacc tcgagttaat taaccaacaa gctcgtcatc gctttgcaga agagcagaga
     3901 ggatatgctc atcgtctaaa gaactaccca ttttattata tattagtcac gatatctata
     3961 acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata acaatataat
     4021 tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca
     4081 cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg
     4141 agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa
     4201 agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt
     4261 taatacgtat aatacatatg attcagctgc attaatgaat cggccaacgc gcggggagag
     4321 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4381 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4441 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4501 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4561 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4621 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4681 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4741 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4801 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4861 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4921 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4981 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5041 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5101 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5161 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5221 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5281 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5341 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5401 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5461 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5521 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5581 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5641 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5701 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5761 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5821 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5881 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5941 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6001 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6061 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6121 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6181 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6241 ttccgcgcac atttccccga aaagtgccac ctgacgtcat ctgttaacat tatacgcgtt
     6301 taaccctaga aagataatca tattgtgacg tacgttaaag ataatcatgc gtaaaattga
     6361 cgcatgtgtt ttatcggtct gtatatcgag gtttatttat taatttgaat agatattaag
     6421 ttttattata tttacactta catactaata ataaattcaa caaacaattt atttatgttt
     6481 atttatttat taaaaaaaaa caaaaactca aaatttcttc tataaagtaa caaaactttt
     6541 aaacattctc tcttttacaa aaataaactt attttgtact ttaaaaacag tcatgttgta
     6601 ttataaaata agtaattagc ttaacctata cataatagaa acaaattata cttattagtc
     6661 agtcagaaac aactttggca catatcaata ttatgctctg ctagcgtttg aacgctgacg
     6721 tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac acccagcgcg
     6781 cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct gcaatatttg
     6841 catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg atttgggaat
     6901 cttataagtt ctgtatgaga ccacttggat ccggcttact aaaagccaga taacagtatg
     6961 cgtatttgcg cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta
     7021 tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta
     7081 tcagttgctc aaggcatata tgatgtcaat atctccggtc tggtaagcac aaccatgcag
     7141 aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct
     7201 gaggtcgccc ggtttattga aatgaacggc tcttttgctg acgagaacag gggctggtga
     7261 aatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt
     7321 acagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg
     7381 tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag
     7441 ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt
     7501 ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg
     7561 aatataaatg tcaggctccc ttatacacag ccagtctgca ggtcgaccat agtgactgga
     7621 tatgttgtgt tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat
     7681 tgatatttat atcattttac gtttctcgtt cagctttctt gtacaaagtg gtgatctagc
     7741 taaaacgcgg cctcgaatcg aattcaaacc cgggcgatc
//
